Figure 3

siRNA-CTGF transfection reduces basal and high glucose-induced CTGF, collagen type I and FN mRNA (a) and protein expression (b, c and d) in HUVSMC. (a) Q-PCR results: Growth-arrested HUVSMCs were transfected with scrambled or CTGF-siRNA plasmids for 24 hours and then exposed to normal glucose (NG) or high glucose (HG) conditions for 24 to 72 hours. CTGF, collagen type I and FN mRNA expression were assayed by Q-PCR. Experiments were performed five times with the similar results (n = 5 in each group). (b) Representative Western blot (top) and values of total CTGF production (means ± SEM of 3 experiments, bottom). Results of total CTGF protein production were obtained from densitometric analysis and expressed as ratio of CTGF/β-actin. (c) Immunocytochemistry staining of collagen type I protein expression in HUVSMCs (top, magnificent of 400×) and integrated optical density (IOD) of the collagen type I staining was measured on the images using the Image-Pro Plus^® software (bottom). Figure shows a representative experiment of 3 performed. (d) Immunocytochemistry staining of fibronectin (FN) protein expression in HUVSMCs (top, magnificent of 400×) and integrated optical density (IOD) of the fibronectin staining was measured on the images using the Image-Pro Plus^® software (bottom). Figure shows a representative experiment of 3 performed. * P < 0.05 vs scrambled siRNA transfection under normal glucose (NG) media condition. # P < 0.05 vs scrambled siRNA transfection under high glucose (HG) media condition. Scrambled siRNA: scrambled siRNA plasmid transfection; siRNA: siRNA-CTGF plasmid transfection; NG: normal glucose; HG: High glucose.